This invention relates to a novel method of in vivo methylation of nucleic acids. In particular, the invention relates to thermophilic Bacillus strains transformed using a plasmid transformation system based on the method of in vivo methylation. The invention can be used to increase ethanol production.
Many bacteria have the ability to ferment simple hexose sugars into a mixture of acidic and pH-neutral products via the process of glycolysis. The glycolytic pathway is universal and comprises a series of enzymatic steps whereby a six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound pyruvate. This process results in the net generation of ATP (biological energy supply) and the reduced cofactor NADH.
Pyruvate is an important intermediary compound of metabolism. Under aerobic conditions (oxygen available), pyruvate is first oxidised to acetyl CoA and then enters the tricarboxylic acid cycle (TCA) which generates synthetic precursors, CO2 and reduced cofactors. The cofactors are then oxidised by donating hydrogen equivalents, via a series of enzymatic steps, to oxygen resulting in the formation of water and ATP. This process of energy formation is known as oxidative phosphorylation.
Under anaerobic conditions (no available oxygen), fermentation occurs in which the degradation products of organic compounds serve as hydrogen donors and acceptors. Excess NADH from glycolysis is oxidised in reactions involving the reduction of organic substrates to products such as lactate and ethanol. In addition, ATP is regenerated from the production of organic acids such as acetate in a process known as substrate level phosphorylation. Therefore, the fermentation products of glycolysis and pyruvate metabolism include a variety of organic acids, alcohols and CO2.
The majority of facultatively anaerobic bacteria do not produce high yields of ethanol either under aerobic or anaerobic conditions. Most faculatative anaerobes metabolise pyruvate aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic acid cycle (TCA).
Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate and acetyl-CoA. Acetyl-CoA is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (AK) with the co-production of ATP, or reduced to ethanol via acetalaldehyde dehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re-oxidised to NAD+ by lactate dehydrogenase (LDH) during the reduction of pyravate to lactate. NADH can also be re-oxidised by AcDH and ADH during the reduction of acetyl-CoA to ethanol but this is a minor reaction in cells with a functional LDH. Theoretical yields of ethanol are therefore not achieved since most acetyl CoA is converted to acetate to regenerate ATP and excess NADH produced during glycolysis is oxidised by LDH.
Ethanologenic organisms, such as Zymomonas mobilis and yeast, are capable of a second type of anaerobic fermentation, commonly referred to as alcoholic fermentation, in which pyruvate is metabolised to acetaldehyde and CO2 by pyruvate decarboxylase (PDC). Acetaldehyde is then reduced to ethanol by ADH regenerating NAD+. Alcoholic fermentation results in the metabolism of 1 molecule of glucose to two molecules of ethanol and two molecules of CO2. DNA which encodes both of these enzymes in Z. mobilis has been isolated, cloned and expressed recombinantly in hosts capable of producing high yields of ethanol via the synthetic route described above.
A key improvement in the production of ethanol using biocatalysts can be achieved if operating temperatures are increased to levels at which the ethanol is conveniently removed in a vaporised form from the fermentation medium. However, at the temperatures envisioned, traditional mesophilic microorganisms, such as yeasts and Z. mobilis, are incapable of growth. This has led researchers to consider the use of thermophilic, ethanologenic bacteria such as Bacillus sp as a functional alternative to traditional mesophilic organisms. See EP-A-0370023.
The use of thermophilic bacteria for ethanol production offers many advantages over traditional processes based upon mesophilic ethanol producers. Such advantages include the ability to ferment a wide range of substrates, utilising both cellobiose and pentose sugars found within the dilute acid hydrolysate of lignocellulose, as well as, the reduction of ethanol inhibition by continuous removal of ethanol from the reaction medium using either a mild vacuum or gas sparging. In this way, the majority of the ethanol produced may be automatically removed in the vapour phase at temperatures above 50xc2x0 C. allowing the production phase to be fed with high sugar concentrations without exceeding the ethanol tolerance of the organism, thereby making the reaction more efficient. The use of thermophilic organisms also provides significant economic savings over traditional process methods based upon lower ethanol separation costs.
The use of facultative anaerobes also provides advantages in allowing a mixed aerobic and anaerobic process. This facilitates the use of by-products of the anaerobic phase to generate further catalytic biomass in the aerobic phase which can then be returned to the anaerobic production phase.
It is possible that organisms which carry out glycolysis or a variant thereof can be engineered to divert as much as 50% of the carbon in a sugar molecule via glycolysis and a synthetic, metabolic pathway which comprises enzymes encoded by heterologous genes. The result is an engineered organism which produces ethanol as its primary fermentation product.
The inventors have produced sporulation deficient variants of a thermophilic, facultatively anaerobic, Gram-positive bacterium which exhibit improved ethanol production-related characteristics. This has been achieved through the development of a plasmid transformation system based on a novel method of in vivo methylation.
The production of recombinant Bacillus sp, engineered to express a heterologous gene, has previously been hampered by a Hae III type restriction system that limited plasmid transformation.
In vivo methylation has been used previously to overcome different restriction problems in other bacteria such as Xanthomonas campestris. For example, De Feyter and Gabriel (De Feyter, R, Gabriel, D. W.) Journal of Bacteriology 173 (1991) (20): 6421-7 have shown that where cosmid libraries of DNA from the bacterium X. campestris were restricted when introduced into strains of Escherichia coli, the use of cloned DNA methylase genes increased the frequency of transfer of foreign genes into X. campestris pv. malracearum. In this instance, restriction was associated with the mcrBC+ gene in E. coli. Restriction was overcome using a plasmid (pUFRO52) encoding the XmaI and XmaIII DNA methylases isolated from X. campestris pv malracearum. Subsequent plasmid transfer from E. coli strains to X. campestris pv. malvacearum by conjugation was significantly enhanced.
Similarly, Mermelstein and Papoutsakis (Mermelstein, L. D, and Papoutsakis, E. T) Appl. Environ. Biology 59(4) (1993) have shown that in vivo methylation in E. coli by B.subtilis phage phi 3TI methyltransferase can be used to protect plasmids from restriction upon transformation of Clostridium acetobutylicum. 
Transformation efficiency in Bacillus strains was initially limited by a HaeIII-type restriction system, previously identified in Bacillus strain LLD-R. Bacillus strain LLD-R possesses a powerful HaeIII type restriction-modification system similar to that found in Haemophilus aegyptius (Zaidi S. H. E. (1991) PhD thesis, Imperial College, London). The HaeIII restriction endonuclease methylates the inner cytosine residues in the recognition site S-GGCC-3 which occurs frequently in the GC rich genome of LLD-R. HaeIII restriction of heterologous plasmid DNA in strain LLD-R presented a major barrier to successful transformation as previous attempts to transform this strain with un-methylated DNA had failed. The inventors partially overcame the problem of heterologous plasmid DNA restriction via the in vitro methylation of plasmid DNA using a commercially available DNA HaeIII methylase. However, in vitro methylation was found to be highly unreliable, costly and time consuming.
Bacillus methanolicus has been transformed using plamid DNA that has been methylated in vitro or in vivo by a host cell having an endogenous dam methylase (Cue etal, Appl. Environ. Microbiology, 63, 1406-1420, 1997).
The inventors have completely overcome the problem of heterologous plasmid DNA restriction using a novel method of in vivo methylation. Complete methylation of heterologous DNA was achieved using an in vivo methylation system incorporating the gene encoding HaeIII methyltransferase from Haemophilus aegyptius. The HaeIII methyltransferase gene was expressed from a compatible plasmid (pMETH) alongside a co-resident shuttle vector (pUBUC) in E. coli. In vivo methylated pUBUC was then used to transform Bacillus strains LLD-R, LN and TN. In vivo methylated pUBUC transformed Bacillus strains LLD-R, LN and TN at significantly higher frequencies than in vitro methylated pUBUC. No transformants were obtained with unmethylated plasmid DNA. Due to the fact that the in vivo methylation system only protects HaeIII restriction sites it is highly specific to the method embodied in the current invention.
Once the problem of heterologous plasmid DNA restriction had been overcome the inventors set out to optimise the plasmid transformation system. The inventors used a method of plasmid transformation based upon electroporation as this had previously been used for transformation of B. stearothermophilus strain K1041, Narumi et al (1992) Biotechnology Techniques 6 No. 1. This method of plasmid transformation was unsuccessful when used with Bacillus strains LLD-R, and TN until the electroporation conditions were optimised and the composition of the regeneration medium was changed. Surprisingly, by changing the electric field from 12.5 kV/cm to 5.0 kV/cm the inventors increased the plasmid transformation efficiency by 10 fold.
The inventors have isolated a transformable sporulation deficient mutant of Bacillus strain LLD-R. Isolation of this mutant removed a further barrier to transformation caused by sporulation, whereby cells readily sporulate after electroporation, inevitably reducing transformation frequency and transformant recovery. The inventors have also developed a shuttle vector which is able to replicate in both E. coli and Bacillus strains, and have developed a novel in vivo plasmid HaeIII methylation system to overcome restriction of heterologous plasmid DNA. The inventors have also developed a reliable and reproducible agar plate medium containing glycerol and pyruvate for aerobic growth of Bacillus strains LLD-R, LN, TN and derivatives thereof. This medium is referred to as TGP. Specifically, the production of organic acids, especially acetate, from sugars in growth media on agar plates has a significant effect upon culture growth and/or viable cell counts. The unpredictable nature of microorganism growth on agar plate media can be explained by the production of organic acids. These acids act to reduce the pH of the growth medium inhibiting cell growth and viability.
The inventors have overcome this problem by developing a growth medium comprising glycerol and/or pyruvate as non-fermentable carbon substrates. The addition of glycerol and/or pyruvate prevents anaerobic fermentation and production of organic acid by-products, thereby reducing the effects of organic acids, such as acetate, on the pH of the growth medium. In this way, viable cell counts obtained on agar plates using the TGP medium have been significantly increased when compared to cell counts obtained on mineral salt mediums and complex mediums containing fermentable sugars such as glucose, sucrose and xylose. The use of TGP medium increases subsequent transformation frequencies, on the basis of higher levels of cell viability, and provides a suitable medium for the short term maintenance of Bacillus strains of the present invention.
These four developments have been combined to produce a novel plasmid transformation system based on in vivo methylation for Bacillus strains LLDR, TN and LN.
Accordingly, a first aspect of the present invention relates to a method of producing a recombinant Escherichia coli comprising in vivo methylation in a host cell by a non-endogenous DNA methylase of a heterologous gene and introducing that in vivo methylated gene into a Bacillus. The heterologous gene is preferably involved in ethanol production. The Bacillus may be a thermophile. Preferably, the Bacillus is selected from B. stearothermophilus; B. calvodex; B. caldotenax; B. thermoglucosidasius; B. coagulans; B. licheniformis; B. thermodenitrificans and B. caldolyticus. The Bacillus may be sporulation deficient.
The heterologous gene may be methylated in any suitable host cell, preferably another bacterium, prior to the introduction of that gene into the Bacillus. For example, the host may be E. coli. 
The host cell contains a non-endogenous DNA methylase enzyme to be used to methylate the heterologous gene. The DNA methylase may be a HaeIII methyltransferase. The use of modified enzymes and synthetic equivalents is within the scope of the invention.
The term xe2x80x9cnon-endogenousxe2x80x9d means that the methylase is heterologous to the host cell i.e. the methylase is not normally produced by the host cell. Preferably the DNA methylase is heterologously expressed in the host cell. For example, the DNA methylase may be expressed from a plasmid in the host cell or from a heterologous methylase gene incorporated into the host cell""s genome. A preferred plasmid is pMETH.
A shuttle vector which is able to replicate in both the host cell and the Bacillus may be used to transfer the methylated heterologous gene between the bacteria. A preferred shuttle vector is pUBUC.
The methylated heterologous gene may be incorporated into the chromosome of the recombinant Bacillus sp.
According to another aspect of the invention, there is provided a method for transforming a Gram-positive bacteria comprising using electroporation at a voltage of about 4.0 to 7.5 kV/cm.
According to another aspect of the invention, there is provided a Bacillus sp which has been transformed with a methylated heterologous gene. The Bacillus may be a thermophile.
Preferred Bacillus include B. stearothermophilus; B. calvodex; B. caldotenax; B. thermoglucosidasius; B. coagulans; B. licheniformis; B. thermodenitrificans and B. caldolyticus. Preferably, the Bacillus is sporulation deficient.
According to another aspect of the invention there is provided a method for the production of a novel agar plate medium for the aerobic growth of Bacillus strains of the invention comprising, addition of a non-fermentable carbon source. The non-fermentable carbon source is preferably glycerol and/or pyruvate.
Aerobic growth of Bacillus strains on the agar medium results in a reduction of the amount of organic acid by-products produced, thereby preventing a reduction in the pH levels of the growth medium, resulting in more consistent and increased cell counts, thereby increasing subsequent transformation frequencies.